RNA electrophoresis!!

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Fri Oct 31 10:33:26 EST 1997


At 14:55 10/31/97 -0800, Han wrote:
>Dear netters,
>
>I encountered a problem in RNA formaldehyde electrophoresis: I added EB 
>in my formaldehyde agarose gel(final concentration 50ug/100ml) and run 
>gel for 2 hours. Amazingly, when I visualize gel under UV lamp I found 
>very high staining background which evenly distributed and composed 
>almost 2/3 area of gel starting from the margin near the wells. 
	** EDITED**

Han:

I do this all the time.  Formaldehyde acts like a "sink" for EtBr.  So for
RNA gels, I only add 1ul of a 10mg/ml stock to agarose.  Volume isn't an
issue, that's 1ul whether it be a 100ml or 150 ml gel.  The only other
place I have EtBr is in the 10X MOPS loading buffer at 1 ug/ ml.    I'm
running the "fast" formaldehyde gels which only uses 3% formaldehyde.
Through trial and error I've worked out these proportions (I guess the term
is "in my hands") such that I can usually photo the gel immedately after
running.  However, as the formadehyde doesn't do well with nitrocellulose,
washing the gel in DDH20 is strongly advised.  I use Hybond N+ for my
northerns, I follow the same rule.  I don't know if formaldehyde, per se,
also affects N+, but I follow my "hunch" and wash it out as well.  If the
gel can soak in DDH20 with a few changes over about two hours, the quality
of the photo gets even better.  

DO not put EtBr in the MOPS running buffer or you will "blow out" gel as it
will be as pink as pink can be.  You'll have to soak it for quite a while
before you can document it.

Hope this helps,
David


=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
Never take life seriously. Nobody gets out alive anyway.
=============================



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